Journal: Oncotarget

Article Title: A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide

doi:

Figure Lengend Snippet: (A) qPCR confirming the overexpression of HOXA9 in hTERT/E6/E7-HOXA9 and U87MG-HOXA9 cells and HOXA9 -silencing in U251-shHOXA9 and GBML18-shHOXA9 cells, comparing to their respective control counterparts. (B) Venn diagram summarizing the number of differentially expressed transcripts in the microarray data in all cell lines. The numbers in each area represent the total number of transcripts within each intersection. (C–F) DAVID was used to query the HOXA9-transcriptome from each cell line (C, hTERT/E6/E7; D, U87MG; E, U251; F, GBML18), in order to identify enriched biological terms on the differentially expressed genes extracted from the microarray data. Statistically significant enriched GO terms are shown for each cell line.

Article Snippet: For HOXA9 silencing , GBML18 and U251 cells were transfected with pGFP-V-RS plasmid containing HOXA9-specific shRNAs or non-effective shRNAs sequences (Origene).

Techniques: Over Expression, Microarray

Journal: Oncotarget

Article Title: A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide

doi:

Figure Lengend Snippet: (A–D) GSEA reveals that the HOXA9 transcriptomes in hTERT/E6/E7 (A) and U251 (C) cells are associated with transcriptional signatures of glioma stem-like cells (Enrichment Score, ES = −0.54, False Discovery Rate, FDR = 0.19; and ES = 0.50, FDR = 0.11, respectively); in U87MG cells (B) , the HOXA9 transcriptome is positively associated with genes that are upregulated in embryonic stem cells (ES = 0.50, FDR < 0.0001); in GBML18 cells (D) , HOXA9 transcriptome is inversely associated with genes upregulated during the neuronal differentiation (ES = 0.75, FDR = 0.21). (E) Representative phase contrast photographs of hTERT/E6/E7 and U87MG neurospheres are shown. (F) Quantification of neurospheres number and size for each cell line ( n = 3; * p < 0.05; *** p < 0.001). (G) Immunofluorescence showing increased Nestin staining in HOXA9-positive cells.

Article Snippet: For HOXA9 silencing , GBML18 and U251 cells were transfected with pGFP-V-RS plasmid containing HOXA9-specific shRNAs or non-effective shRNAs sequences (Origene).

Techniques: Immunofluorescence, Staining

Journal: Oncotarget

Article Title: A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide

doi:

Figure Lengend Snippet: (A and B) Determination of the half inhibitory concentration (IC 50 ) values after 6 days of temozolomide (TMZ) treatment in HOXA9 -positive or HOXA9 -negative hTERT/E6/E7 and U87MG (A), and HOXA9 -silenced or control U251 and GBML18 (B) cell lines. (C–F) Cell viability trypan blue assays in HOXA9 -negative/low or HOXA9 –positive/high hTERT/E6/E7 (C), U87MG (D), U251 (E) and GBML18 (F) cells, exposed to temozolomide or vehicle. (G) Cell death was evaluated by annexin V staining followed by flow cytometry in HOXA9 -positive/high and HOXA9 -negative/low hTERT/E6/E7, U87MG, U251 and GBML18 cell lines, both in basal conditions and after exposure to temozolomide (TMZ). HOXA9 expression decreases cell death of all GBM cell models, both in basal conditions and after TMZ treatment, except in basal conditions for U251 cell line. (H) Cell invasion in the same cell lines, both in basal conditions and after exposure to TMZ. HOXA9 increases the invasion of hTERT/E6/E7, U87MG, and GBML18 cells. U251 cells did not show significant differences in invasion profiles due to HOXA9 levels or TMZ treatment. Results are representative of at least three independent experiments, performed in triplicates (data points represent mean ± SEM). Statistical differences were calculated by Student's t -test (panels A, B, G, H) and two-way ANOVA (panels C–F) (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: For HOXA9 silencing , GBML18 and U251 cells were transfected with pGFP-V-RS plasmid containing HOXA9-specific shRNAs or non-effective shRNAs sequences (Origene).

Techniques: Concentration Assay, Staining, Flow Cytometry, Expressing

Journal: Cancers

Article Title: Paliperidone Inhibits Glioblastoma Growth in Mouse Brain Tumor Model and Reduces PD-L1 Expression

doi: 10.3390/cancers13174357

Figure Lengend Snippet: DRD2 involved in the PD-L1 expression in GBM cells in the GBM-macrophage co-culture system. ( A ) U87-GFP and U251-GFP cells were co-cultured with the THP-1 differentiated macrophage for 48 h. PD-L1 expression was determined by flow cytometry analysis. ( B ) U87-GFP and U251-GFP cells were treated with paliperidone (20 μM, PAL) for 30 min, and cells were then co-cultured with THP-1 macrophages (HM) after wash-out of paliperidone for 48 h. PD-L1 expression on GBM was determined by flow cytometry analysis. * p < 0.05 compared with GBM alone group. # p < 0.05 compared with THP-1 group. ( C ) THP-1 macrophages (HM) were treated with paliperidone (20 μM, PAL) for 30 min, washed out for 48 h, and then co-cultured with U87-GFP and U251-GFP cells. PD-L1 expression on cell surface of HM was determined by flow cytometry analysis. * p < 0.05 compared with THP-1 alone group. # p < 0.05 compared with U87 or U251 groups ( n = 3–4).

Article Snippet: Human glioma U251 cells were purchased from the (JCRB NO. IFO50288, Tokyo, Japan).

Techniques: Expressing, Co-Culture Assay, Cell Culture, Flow Cytometry

Journal: Cancers

Article Title: Paliperidone Inhibits Glioblastoma Growth in Mouse Brain Tumor Model and Reduces PD-L1 Expression

doi: 10.3390/cancers13174357

Figure Lengend Snippet: GBM primed macrophage potentiates PD-L1 expression in GBMs. Human U87 ( A , C , E ) and U251 ( B , D , F ) GBM were incubated with GBM cultured medium, THP-1 cultured medium, HM CM, or HM/GCM CM for 48 h. PD-L1 expression was determined by flow cytometry analysis ( A , B ), Western blot ( C and D ), and real time-PCR ( E , F ). * p < 0.05 compared with GBM alone group. # p < 0.05 compared with HM CM group. ( n = 3–4).

Article Snippet: Human glioma U251 cells were purchased from the (JCRB NO. IFO50288, Tokyo, Japan).

Techniques: Expressing, Incubation, Cell Culture, Flow Cytometry, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: Paliperidone Inhibits Glioblastoma Growth in Mouse Brain Tumor Model and Reduces PD-L1 Expression

doi: 10.3390/cancers13174357

Figure Lengend Snippet: DRD2 is involved in the interaction of GBM-macrophage-induced PD-L1 expression in GBM. ( A , C , E ) Human U251 GBMs were incubated with human macrophage (HM) cultured medium, HM CM or HM/GCM CM, and then treated with paliperidone (20 μM, PAL), risperidone (20 μM, RS), or L741626 (1 μM) for 48 h. ( B , D , F ) Mouse ALTS1C1 GBMs were incubated with BMDMs cultured medium, BMDMs CM or BMDM/ACM CM, and then treated with or without PAL (20 μM), risperidone (20 μM, RS), or L741626 (1 μM) for 48 h. PD-L1 expression on the cell surface was determined by flow cytometry analysis ( A – D ) and Western blot ( E , F ). * p < 0.05 compared with HM medium group. # p < 0.05 compared with HM/GCM CM group. ( n = 3–4) ( G , H ) mRNA levels of PD-L1 and DRD2 were determined by real-time PCR. * p < 0.05 compared with HM medium or BMDMs medium groups. # p < 0.05 compared with HM/GCM CM or BMDM/ACM CM groups ( n = 3).

Article Snippet: Human glioma U251 cells were purchased from the (JCRB NO. IFO50288, Tokyo, Japan).

Techniques: Expressing, Incubation, Cell Culture, Flow Cytometry, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: Paliperidone Inhibits Glioblastoma Growth in Mouse Brain Tumor Model and Reduces PD-L1 Expression

doi: 10.3390/cancers13174357

Figure Lengend Snippet: ERK and STAT3 signaling pathways are involved in the interaction of GBM-macrophage-induced PD-L1 expression in GBMs. Human U251 ( A ) and mouse ALTS1C1 ( B ) GBM were incubated with HM/GCM CM or BMDM/ACM CM for the indicated time periods (5, 10, 30, 60, or 120 min). Human U251 ( C ) and mouse ALTS1C1 ( D ) GBM were treated with PAL (20 μM), RS (20 μM), or L741626 (1 μM) and incubated with HM/GCM CM or BMDM/ACM CM. Phosphorylated-STAT3 and phosphorylated ERK expressions were determined by using Western blot analysis. Human U251 ( E ) and mouse ALTS1C1 ( F ) GBM were treated with S31-201 (30 μM) or U0126 (1 μM) and incubated with HM/GCM CM or BMDM/ACM CM for 48 h. PD-L1 expression was determined by using Western blot analysis. ( n = 3–4).

Article Snippet: Human glioma U251 cells were purchased from the (JCRB NO. IFO50288, Tokyo, Japan).

Techniques: Protein-Protein interactions, Expressing, Incubation, Western Blot

Journal: Cell Death & Disease

Article Title: The CDK inhibitor AT7519 inhibits human glioblastoma cell growth by inducing apoptosis, pyroptosis and cell cycle arrest

doi: 10.1038/s41419-022-05528-8

Figure Lengend Snippet: A Process for high-throughput drug screening. B , C U87MG, U251, GBM60, and GBM38 cell viability was determined using a CCK-8 assay after treatment with various concentrations of AT7519. * P < 0.05, *** P < 0.01, *** P < 0.001, and **** P < 0.0001 compared with the control using one-way ANOVA followed by Dunnett’s multiple test. D U87MG and U251 cell viability was determined using a CCK-8 assay after treatment with 0.4 µM AT7519 for 6, 12, 24, 48, and 60 h. * P < 0.05, **** P < 0.0001 compared with the control using one-way ANOVA followed by Dunnett’s multiple test. E Colony formation assays to verify the effect of AT7519 on glioblastoma cell proliferation. * P < 0.05, *** P < 0.001, and **** P < 0.0001 based on one-way ANOVA followed by Dunnett’s multiple test. F , G The level of DNA synthesis of U87MG and U251 cells was determined using an EdU assay after treatment with increasing concentrations of AT7519. The nuclei were stained with Hoechst (blue), and the proliferating cells were stained with EdU (yellow). * P < 0.05, *** P < 0.001 and **** P < 0.0001 based on one-way ANOVA followed by Dunnett’s multiple test. The results are presented as the mean ± SD from three independent experiments.

Article Snippet: Human glioblastoma cell lines (U87MG and U251) were purchased from ATCC.

Techniques: High Throughput Screening Assay, Drug discovery, CCK-8 Assay, Control, DNA Synthesis, EdU Assay, Staining

Journal: Cell Death & Disease

Article Title: The CDK inhibitor AT7519 inhibits human glioblastoma cell growth by inducing apoptosis, pyroptosis and cell cycle arrest

doi: 10.1038/s41419-022-05528-8

Figure Lengend Snippet: A U87MG and U251 cells were treated with AT7519 and morphological features of pyroptosis in SEM (red arrows, membrane pore-forming). B After U87MG and U251 cells were treated with AT7519 for 48 h, cytotoxicity was detected by lactate dehydrogenase (LDH) released into the cell culture medium. **** P < 0.0001 by Student’s t -test. C , D Full-length GSDME (GSDME-FL) and N-terminal GSDME (GSDME-N) were detected in glioblastoma cells after treatment with AT7519 for 48 h by western blot analysis. E , F U87MG and U251 cells were pretreated with Z-VAD-FMK for 2 h and then treated with AT7519 for 48 h. Cytotoxicity was detected by LDH release assay. *** P < 0.001 and **** P < 0.0001 as assessed by Student’s t -test. The apoptosis marker cleaved PARP and pyroptosis marker GSDME-N were detected by western blot. G U87MG and U251 cells were treated with Z-DEVD-FMK combined with AT7519 for 48 h, and western blot analysis of cleaved caspase-3, GSDME-FL and GSDME-N proteins was performed.

Article Snippet: Human glioblastoma cell lines (U87MG and U251) were purchased from ATCC.

Techniques: Membrane, Cell Culture, Western Blot, Lactate Dehydrogenase Assay, Marker

Journal: Cell Death & Disease

Article Title: The CDK inhibitor AT7519 inhibits human glioblastoma cell growth by inducing apoptosis, pyroptosis and cell cycle arrest

doi: 10.1038/s41419-022-05528-8

Figure Lengend Snippet: A Image of the subcutaneous xenograft tumors formed in nude mouse models. B Tumor volumes were measured and calculated every week. **** P < 0.0001 as assessed by two-way ANOVA followed by Sidak’s multiple test. C Tumors were excised and weighed at the end of the experiment. *** P < 0.001 by Student’s t -test. D Body weight of nude mice during administration of AT7519. E Representative H&E-stained images of the intracranial xenograft model in the AT7519 treatment group and control group. * p < 0.05 as assessed by Student’s t -test. F Western blot assay of the apoptosis, pyroptosis, and cell cycle-related key protein expression levels in tumor tissue. G Schematic model of the antitumor mechanism of AT7519 in glioblastoma cells.

Article Snippet: Human glioblastoma cell lines (U87MG and U251) were purchased from ATCC.

Techniques: Staining, Control, Western Blot, Expressing

Journal: Journal of Biological Engineering

Article Title: Advanced biomanufacturing and evaluation of adeno-associated virus

doi: 10.1186/s13036-024-00409-4

Figure Lengend Snippet: Confocal microscope demonstrating high transduction of AAV, revealed by co-localization of green GFP (U251 cells), blue DAPI (nucleus), and red Sulfo-cyanine 5.5 (AAV). MOI = 5,000

Article Snippet: The human glioblastoma cell line U251 (MilliporeSigma, Burlington, MA, USA) was cultivated in DMEM/F12 medium with 10% fetal bovine serum, 4 mM L-glutamine, 1% non-essential amino acids (NEAA), 1 mM sodium pyruvate, and 1% penicillin/streptomycin (100 IU/100 μg/mL) in T-75 flask at 37 °C and 5% CO 2 [ ].

Techniques: Microscopy, Transduction

Journal: Journal of Biological Engineering

Article Title: Advanced biomanufacturing and evaluation of adeno-associated virus

doi: 10.1186/s13036-024-00409-4

Figure Lengend Snippet: Evaluations of functional gene expression. A Live-animal IVIS imaging showed high in vivo expression of AAV-delivered gene. About 0.5 × 10 6 U251 cells were intracranially injected to NSG mice using stereotactic instrument to develop glioblastoma xenografted models. AAV (1 × 10 11 vg) and ViviRen (3.7 μg) were injected. B In vitro AAV gene expression is dosage (multiplicity of infection, MOI)-dependent. C AAV gene expression correlates to MOI, as measured by i3x plate reader

Article Snippet: The human glioblastoma cell line U251 (MilliporeSigma, Burlington, MA, USA) was cultivated in DMEM/F12 medium with 10% fetal bovine serum, 4 mM L-glutamine, 1% non-essential amino acids (NEAA), 1 mM sodium pyruvate, and 1% penicillin/streptomycin (100 IU/100 μg/mL) in T-75 flask at 37 °C and 5% CO 2 [ ].

Techniques: Functional Assay, Gene Expression, Imaging, In Vivo, Expressing, Injection, In Vitro, Infection

Journal: Cancer Cell International

Article Title: Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

doi: 10.1186/s12935-018-0710-0

Figure Lengend Snippet: mTOR activity and metabolic differences in U251, U87 and U373-U human glioma cells. a mTOR activity related proteins and other metabolic enzyme expressions characterise and show some individual differences in the studied human glioma cell lines—representative figures of Western blot results; b the enzyme expression profiles could correlate to mTOR inhibitor sensitivity of glioma cells (rapamycin—Rapa 50 ng/mL; NVP-BEZ235—BEZ 1 µM; PP242 1 µM for 72-h treatments), which were monitored by Alamar Blue and SRB proliferation tests—the cell proliferation of untreated controls was considered 100%; rapamycin inhibited the proliferation in all studied cells, significantly; Significant differences compared to rapamycin were labelled by *p < 0.05

Article Snippet: U251 (ECACC-09063001, characteristic glioma cell specific mutations in PTEN, NF-1, p53, MSH2), U87 (ATCC-HTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and Notch-2), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: Activity Assay, Western Blot, Expressing

Journal: Cancer Cell International

Article Title: Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

doi: 10.1186/s12935-018-0710-0

Figure Lengend Snippet: The anti-proliferative effects and alterations in mTOR activity and other metabolism related protein levels in response to mTORI and temozolomide treatments in human glioma cell lines. a mTOR inhibitors (rapamycin—Rapa 50 ng/mL; NVP-BEZ235—BEZ 1 µM; PP242 1 µM) and temozolomide (TMZ 100 µM) 72-h combination treatments effectively inhibited the proliferation of U251, U87 and U373-U cells—Alamar Blue and SRB proliferation test results, the cell proliferation of untreated controls were considered 100% (temozolomide slightly reduced the proliferation and had no significant growth inhibitory effect on U251, mTORIs had similar effects as in Fig. a, and all temozolomide + mTORI combined treatments had significant anti-proliferative effects, p < 0.05; the additive (A) or synergistic (S) effects of inhibitor combinations were given based on CI calculation, SD was added); b altered expressions of mTORC1 and C2 activity related proteins and other metabolic enzymes were also shown after different mono-treatments (results of representative Western blots after 72-h treatments)

Article Snippet: U251 (ECACC-09063001, characteristic glioma cell specific mutations in PTEN, NF-1, p53, MSH2), U87 (ATCC-HTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and Notch-2), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: Activity Assay, Western Blot

Journal: Cancer Cell International

Article Title: Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

doi: 10.1186/s12935-018-0710-0

Figure Lengend Snippet: Metabolic drugs induced proliferative and protein expression profile changes in human glioma cells. a Doxycycline (Doxy 10 µM), etomoxir (Etom 50 µM), chloroquine (Chl 50 µM) 72-h treatments have slight growth inhibitory effects in U251, U87 and U373-U (Alamar Blue proliferation test data; *p < 0.05); b ACSS2, FASN, CPT1a and p-(Ser473)-Akt protein expressions were influenced by rapamycin, doxycycline, etomoxir and chloroquine after 72-h mono-treatments; c FASN, β-F1-ATPase, p-S6, p-(Ser473)-Akt and CPT1a protein expression compared to the untreated controls after applying two-drug combinations in U251 glioma cells (50 ng/mL rapamycin + 100 µM temozolomide—Rapa + TMZ; 50 ng/mL rapamycin + 10 µM doxycycline—Rapa + Doxy; 100 µM temozolomide + 10 µM doxycycline—TMZ + Doxy (Western blot results)—representative Western blot figures

Article Snippet: U251 (ECACC-09063001, characteristic glioma cell specific mutations in PTEN, NF-1, p53, MSH2), U87 (ATCC-HTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and Notch-2), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: Expressing, Western Blot

Journal: Cancer Cell International

Article Title: Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

doi: 10.1186/s12935-018-0710-0

Figure Lengend Snippet: Rapamycin and temozolomide combined with other metabolic inhibitors have different in vitro anti-proliferative effects in human glioma cell lines. Alamar Blue test results after 72-h different two-drug combined in vitro treatments in U251 ( a ), U87 ( b ) and U373-U ( c ) glioma cells (rapamycin—Rapa 50 ng/mL, temozolomide—TMZ 100 µM, doxycycline—Doxy 10 µM; etomoxir—Etom 50 µM; chloroquine—Chl 50 µM). The additive (A) or synergistic (S) effects of combinations were given based on CI calculation, SD was added, the anti-proliferative effects were significant (p < 0.05) in almost all combined treatments compared to untreated cultures except for doxycycline + etomoxir and temozolomide + etomoxir treatments

Article Snippet: U251 (ECACC-09063001, characteristic glioma cell specific mutations in PTEN, NF-1, p53, MSH2), U87 (ATCC-HTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and Notch-2), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: In Vitro